CSUPERB ABSTRACTS

Resveratrol (3,5,4’-trihydroxystilbene) is a natural compound primarily found in red grape skins, red wine, blueberries, and several plants like eucalyptus. Resveratrol is a potent antioxidant and inflammatory suppressant that has been shown to extend the lifespan and minimize aging in C. elegans, Drosophila, fish, and mice. However, very little research has been conducted using human cells. We propose to study the potential effect of short- and long-term resveratrol treatment on human adipogenesis, osteogenesis and adult stem cell self-renewal by using human mesenchymal stem cells (hMSCs) as our in vitro cell model. Human mesenchymal stem cells (hMSCs) are multipotent stem cells that are present in the bone marrow and adipose tissue. They have the potential to differentiate into many mature cell types, including but not limited to adipocytes, osteoblasts, chrondrocytes, myocytes, islet cells, and neurons upon receiving appropriate external stimuli. Using hMSCs in this study will help us understand how resveratrol affects humans in vivo.Our preliminary data demonstrated that 1uM or 5uM resveratrol inhibited adipogenic differentiation of hMSCs. These hMSCs also experienced a 3-4 day delay in mature fat cell formation and produced fewer fat cells than treatments without resveratrol. Resveratrol treatments less than 1uM (0.01uM and 0.1uM) experienced the same delay in mature fat cell formation but produced similar amounts of mature fat cells compared to control treatments without resveratrol. In addition, 0.1uM and 1uM resveratrol enhanced osteogenic differentiation of hMSCs. Lower resveratrol concentration (0.01uM) appeared to have no effect on osteogenic differentiation, however higher concentrations (5uM) appeared to slightly inhibit osteogenic differentiation. Resveratrol treatments greater than 10uM were found to be toxic to hMSCs. Currently we are investigating the potential long term effect of resveratrol treatment on the self-renewal and differentiation capacity of hMSCs.In the future studies we will examine potential expression changes of known adipogenic and osteogenic markers in response to resveratrol treatment under conditions in which resveratrol demonstrates the strongest morphological effect on the cells. We will also use agonist/antagonist studies to investigate the potential effect of resveratrol on signaling pathways known to play important roles in hMSCs differentiation.

Biofilm Formation on Experimental Alloys for Human Prosthetics

Student: Jessamine QuijanoResearch Advisor: Steve Alas

Human prosthetics are being utilized more frequently as the populations that require their use expand. These cohorts include an increasing elderly population, American soldiers returning with war injuries, and members of the general public who suffer serious injury from accidents. The development of modern human prosthetics has resulted in more biocompatible implants, but they lack longevity due to prosthetic loosening that is caused by metal corrosion or chronic infection. One major area of concern is the formation of antibiotic resistant biofilm on the prosthetic surface during infections. Using Pseudomonas aeruginosa, a bacteria that commonly creates biofilm formation in patient implants, I investigated the biocompatibility of novel titanium alloys with regard to their susceptibility to biofilm propagation. I examined stainless steel (SS), commercially pure titanium (Ti), Ti-6Al-4V (Ti64) and 3 novel titanium alloys that contained either 0.05% Boron, 0.4% Boron, or 1.0% Boron. To determine the amount of biofilm growth, I performed crystal violet staining. Ideal growth conditions were obtained using flasks with 50mL of TSB media for 24 hours at 37˚C or using a biofilm reactor with 350mL TSB media, and allowing incubation for 48 hours at 37˚C. My preliminary results show that the Ti64 alloy permits less biofilm formation than SS and Ti in flask conditions by P. aeruginosa, using crystal violet staining. Using the biofilm reactor, I determined that P. aeruginosa resulted in less biofilm formation on Ti64 than SS, Ti, and the other alloys tested. In order to characterize the viability of the cells comprising the biofilm, I used the BacLight staining procedure and fluorescent microscopy. I determined that the biofilm created contained a large percentage of dead cells. However, more analysis must be done to quantify biofilm viability grown on the various novel alloys, as this is an indicator of prosthetic longevity. At this stage, my data indicates that the Ti-6Al-4V alloy may allow less biofilm formation than traditional metals and, thus, may be a better alternative to stainless steel and pure titanium as a prosthetic biometal.

Introduction: Influenza virus (INFV), which infects three to five million people every year, is an enveloped, single stranded RNA virus. The most susceptible population to seasonal INFV are immunocompromised individuals including elderly, pregnant women, and children up to 5 years old. Given reports that females produce a stronger immune response than males, the present experiment was done to investigate if there was a difference between infection severity in male and female mice challenged with H1N1 influenza virus and if the antibody response to viral challenge differed between sexes.Materials and Methods: Sedated Swiss Webster male and female mice were challenged intranasally with 40l (1:4-1:512 dilutions) of mouse-passaged H1N1 (A/PR/8/34) viral stock. Mice were monitored daily for 5-6 days for weight loss and disease signs on a scale of 0-4. Day 4 or 5 post-challenge, some groups of mice (n=5-6/group) were sacrificed with their lungs and blood collected for determination of foci of infection and hemagglutination inhibition titer, respectively. In the foci assay, lungs were weighed, homogenized, serially diluted, mixed with MDCK cells (7×106cells/well), and incubated for 48h. Anti-NP (viral nucleoprotein) antibody was used to histochemically visualize the number of infected cells (foci of infection) in the MDCK monolayer. Results: Initial studies showed that to obtain comparable infection for female and male mice, we needed to administer a higher concentration of H1N1 virus (1:4 dilution) for females than for males (1:16 dilution) indicating that the females were more resistant to infection. Day of death for both sexes given comparable viral challenge doses was day 6. Disease severity at these doses was similar. In a subsequent experiment using these doses, the lungs of male and female infected mice were collected and the foci assay demonstrated that male and female mice were equally infected (8.0×104/g lungs vs 4.7×104/g lungs, respectively; p=0.0262). This was reflected in similar HAI titers for the male and female mice (1.0×103 HAIU/mL, p=1.0000).Conclusions: The data showed that the males were more susceptible to the H1N1 infection than the females since they needed to be challenged with a lower concentration of virus to produce a similar degree of infection as the females. Future studies with candidate H1N1 vaccines will require testing in male and female mice that are challenged with viral doses that produce comparable infection.

Key words: H1N1 virus, sexes, susceptibility

Sulfhydryl Modification of the γ-Aminobutyric Acid Transporter 1 (GAT1) at a Single Solvent-Exposed Cysteine Residue

Regulation of inhibitory -aminobutyric acid (GABA) signaling in the brain is carried out by high-affinity GABA transporters (GATs) found in the plasma membrane of neurons and glia. The objective of the present study was to utilize extracellulary-exposed cysteine residues of GAT1 to label the transporter in the plasma membrane. GAT1 contains fourteen cysteine residues of which only three (C74, C164, and C173) are predicted to be exposed to the extracellular fluid. Cysteine 74 is in the first transmembrane domain near the membrane/extracellular fluid interface. Cysteines 164 and 173 are located in the extracellular loop connecting transmembrane domains three and four, and are thought to form a disulfide bridge under most conditions. We expressed human GAT1 in Xenopus laevis oocytes and used electrophysiological methods to assay transporter function before and after sulfhydryl modification with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), a membrane-impermeant alkylating reagent. In the presence of NaCl, transporter exposure to MTSET (1 mM for 5 min.) inhibited GAT1-mediated transport by 48%. Increasing the concentration of MTSET to 2.5 mM (5 min.) inhibited transport by 52%, and increasing the incubation time with MTSET to 20 min. (1 mM) inhibited transport by 66%. Loss of transporter function caused by alkylation was reversed with the reducing reagent dithiothreitol. Importantly, MTSET treatment had no functional effect on the mutant GAT1 C74A, suggesting that C74 is the only functionally-significant cysteine residue that is accessible from the extracellular fluid. As GAT1-mediated GABA transport is Na+- and Cl–-coupled, we investigated the degree of MTSET labeling when Na+ and/or Cl– was isosmotically replaced with various cations/anions in the labeling buffer. The largest effect was seen when valproate replaced Cl– in the labeling buffer; 90% inhibition (1 mM MTSET for 5 min.). Time-dependence of MTSET labeling in the presence of Na-valproate demonstrated transport inhibition of up to ~95% (1 mM MTSET for 20 min.). These results suggest that C74 is partially exposed to the extracellular fluid, and that cation/anion interactions with the transporter alter C74 accessibility to the extracellular fluid. Future studies will use radiolabeled MTSET in order to quantify GAT1 copies in the plasma membrane. (Supported by NIH grant SC1GM086344 awarded to S.E.)

SCCUR ABSTRACTS

The goal of this research is to investigate bactericidal properties of magnesium oxide (MgO) xerogels and aerogels in new structures such as pure MgO thin films and MgO-polylactic acid polymer composite films for use in food safety. In this work, the sol-gel method is used to synthesize amorphous MgO xerogels (ambiently dried) and aerogels (super-critically dried) that have a highly porous structure, giving them a greater surface area and larger concentration of structural defects than crystalline MgO. Since MgO surface defects are where the biocidal activity takes place, there should be a correlation between bactericidal activity and the surface structures obtained by the different preparation methods. Preliminary results show MgO has been effective against Escherichia coli and Staphylococcus aureus cultures.

Student: Jessamine Quijano, Research Advisor: Steve AlasThe development of modern human prosthetics has resulted in more biocompatible implants. However, they lack longevity due to prosthetic loosening caused by metal corrosion or chronic infection. Of concern is the formation of antibiotic resistant biofilm on prosthetic surfaces. Using Pseudomonas aeruginosa, which commonly creates biofilm on patient implants, I investigated novel titanium alloys and their susceptibility to biofilm propagation. I examined stainless steel, commercially pure titanium, Ti-6Al-4V and 3 novel titanium-boron alloys. I cultured Pseudomonas in a biofilm reactor containing samples of each biometal. Utilizing crystal violet staining, my preliminary results show that the Ti-6Al-4V alloy permits less biofilm formation than all other tested metals, indicating that it may be a better alternative to traditional prosthetic biometals.

Studying secreted proteins in pathogenic bacteria aids in vaccine development by providing insight into mechanisms of bacterial infection. Our model for studying bacteria secretion is Tsh, of pathogenic E.coli. In our research, we determine surface localization of Tsh by performing immunofluorescence microscopy with anti-Tsh antibody. The aim of this research is to develop a more efficient protocol for determining the surface localization of Tsh in bacteria by fluorescence microscopy. Here we present a comparison between the original procedure and a new one we developed. The adapted protocol offers several advantages over the original: better visualization of proteins and greater ease of preparation.

Other Abstracts

The goal of this research is to investigate bactericidal properties of magnesium oxide (MgO) xerogels and aerogels in new structures such as pure MgO thin films and MgO-polylactic acid polymer composite films for use in food safety. Experiments suggest that these bactericidal properties result from surface defects, such as unsaturated surface oxygen ions. It is thought that contact with the MgO surface inhibits bacterial growth by interrupting vital bacterial cell functions. In this work, the sol-gel method is used to synthesize MgO xerogels and aerogels that have a highly and amorphous porous surface, giving them a greater surface area and larger concentration of surface defects than crystalline MgO. Of the three types of materials studied, crystalline MgO has the lowest surface area, ambiently dried xerogels have a higher surface area, and supercritically dried aerogels have the highest surface area of the three. Current results through the plate count method show with 5% MgO aerogel concentration there is a 99% reduction in Escherichia coli colonies after an incubation time of one hour. The aforementioned results pertaining to the inhibition of bacterial colonies through the use of MgO, imply MgO aerogel is a formidable material for the production of antibacterial polymers for use in food safety.

Population structure of Navanax based on nuclear and mitochondrial gene data

Student: Elysse Ranette GatdulaResearch Advisor: Dr.Valdez

Along the eastern Pacific several genera of the family Aglajidae can be found. Of particular interest are species belonging to the genus Navanax, which includes N. aenigmaticus, N. inermis, and N. polyalphos. While N. inermis and N. polyalphos are restricted to the eastern Pacific, populations of N. aenigmaticus are found in the eastern Pacific, western Atlantic, and eastern Atlantic. These species were described using morphological characters, such as color and the anatomy of the digestive and reproductive systems. However, members of these species exhibit wide color variation and it is not known if color forms are genetically distinct. What also remains unknown is if the three isolated populations of N. aenigmaticus are genetically distinct despite a similar morphology. The primary use of morphological characters to describe new species has led to questions regarding the validity of some species. New species are sometimes described solely on the basis of some color forms and fail to identify the color range within species. To address the validity of the three species of Navanax, two molecular markers were employed to construct a molecular phylogeny of the genus. The mitochondrial 16S ribosomal gene has both highly conserved and highly variable regions; the variable regions are loops regions which are subject to relatively high mutation rates. The nuclear H3 histone protein-coding gene is highly conserved, but is subject to silent mutations accumulated at a much slower rate than those seen in the 16S gene. Using the molecular phylogeny, we can determine the validity, the color variation, and the geographic structure for each species. The phylogenies suggest the synonymization of two species of Navanax and the reinstatement of another species.

The atmosphere is the largest pool of terrestrial nitrogen. The vast majority of life on earth is unable to directly incorporated atmospheric nitrogen for biological processes. Biological nitrogen fixation is the reduction of atmospheric N2 molecules to biologically incorporable nitrogen compounds, such as ammonia, mediated by the symbiotic interaction of plants known as legumes and soil prokaryotes collectively known as Rhizobia. This interaction begins with a complex molecular dialogue between the plant and the prokaryotic partner. The plant’s root system secrets phenolic compounds known as flavonoids that trigger the expression of Nod genes in the prokaryote resulting in the translation of Nod factor molecules. The perception of Nod factors by the plant allow it to transcribe and translate plant genes known as Nodulins which are responsible for the physiological and developmental changes in the plant necessary for a successful symbiotic interaction. The interaction culminates in the infection of nodules, novel plant root system structures that serve as the active site of atmospheric nitrogen reduction, by the prokaryote. Recent studies suggest that plants might be capable of perceiving Nod factor signal molecules at a much earlier developmental stage. The purpose of our work is to explore the possibility of Nod factor perception by seeds.

Abstract 2:

Plant-Microbe Interaction project: Construction and application analysis of the wide range red fluorescence conferring plasmid PHC60mCherry, a novel tool with applications in the study of the Rhizobia-Legume symbiosis with multiple fluorescence- tagged microscopy

Here we present the construction of a wide range fluorescent protein expression vector that is compatible with other commonly used fluorescence conferring vectors in gram negative bacteria. Cloning the red autofluorescent protein “mCherry” in the wide range vector pHC60 has produced a useful tool for various molecular applications such as multiple fluorescent tagged microscopies. The destination vector is capable of constitutively expressing genes in various genera (Rhizobium, Sinorhizobium, ect.). Additionally, its toxin-antitoxin origin of replication makes it highly compatible with plasmid vectors with pBBR origins. The pHC60 vector originally contains a GFP sequence under its constitutive promoter. The GFP coding sequence was liberated from the plasmid by a digestion reaction with the restriction enzyme BglII. A subsequent double digestion with the restriction enzymes KpnI and XhoI allowed the pHC60 plasmid without GFP to become linear. The mCherry coding sequence was also liberated from the pJPO11 pCAGGS Chop-mCherry-WPR cassette by digestion with the same restriction enzymes. Compatible ends then facilitated a straight forward ligation reaction of the mCherry fragment with the linear pHC60V (GFP free) target vector. Following PCH60mCherry plasmid sequencing, we observed, by fluorescent microscopy, Rhizobium leguminosarum biovar viciae labeled with the new pHC60mCherry plasmid in free-living and symbiotically associated states.

American Chemical Society

Determination of residue-specific binding interactions between retrocyclin and the heptad repeat region of gp41

Student: Michael PeraltaResearch Advisor: Dr.Shantanu Sharma

The -defensin peptide retrocyclin(RC) binds the heptad repeat(HR) region of HIV-1 glycoprotein 41 (gp41) inhibiting cell entry, thus preventing new infections. In our study, complementary in silico and in vitro methods are utilized to predict and then structurally characterize the RC-HR binding site(s). We employed a Monte Carlo method-docking algorithm to exhaustively sample possible conformations of the docked complex. The algorithm evaluated the conformational energy (score) of generated structures, enabling us to select 3 low scoring and distinct binding modes from the initial 30,000. These structures were subjected to 50ns of structural and energetic molecular dynamics refinement and residues contributing to binding stability were mutated in silico. A library of RC analogs are being synthesized and binding affinities measured using SPR. These experimental results will be compared with computational predictions. By understanding the specific interactions that confer and disrupt structural stability, we can ultimately design even more effective RC variants.

Bacterial spores are the most resistant forms of life and have been a major threat to public health and food safety. Plasma is a novel sterilization method that leaves no chemical residue and works at low temperatures. This study used helium-based, non-thermal, gas discharge plasma to treat spores of Bacillus subtilis, Bacillus pumilis, and Clostridium sporogenes in order to test sporocidal and bactericidal effects. Previous studies revealed that leakage of dipicolinic acid (DPA) occurred after exposure to plasma, and that D-values for Bacillus and Clostridium species ranged from 2 to 5 minutes after exposure. Optical emission spectroscopy revealed that the dominant species of plasma are strong oxidizing agents, such as free radicals and UV photons. Leakage of spore contents indicates that exposure to oxidizing agents and free radicals may lead to spore coat damage responsible for spore inactivation. This study investigated the surface damage caused by gas-discharge plasma by visualization of treated and untreated spores using scanning electron microscopy (SEM). The result should aid in our understanding of the mechanism of killing by plasma.

Abstract 2

DNA translocation by the molecular motor SpoIIIE

Bacillus subtilis is a gram positive bacteria with the ability to form spores in order to withstand harsh environmental conditions. Molecular motors convert energy into mechanical work. SpoIIIE (a member of the FtsK/SpoIIIE family of ATP dependent DNA transporter proteins) is a molecular motor that moves DNA between cellular compartments mediating proper chromosome segregation during B. subtilis sporulation. SpoIIIE assembles as hexamers in the septum of the sporulating bacteria though the exact mechanochemical coordination among subunits is not clear. We sought out to test whether ATP hydrolysis of the subunits involves concerted, sequential, or random firing. The effects on the mechanochemical properties of the ring were studied by introducing a point mutation at the ATP binding site, creating inactive subunits. Different concentrations of these inactive subunits, creating hexamers with different configurations, were compared regarding ATP hydrolysis activity and their ability to displace a DNA triplex substrate in bulk. Malachite green dye method was used to measure the phosphate released during ATP hydrolysis by both wildtype SpoIIIE and mutant SpoIIIE (R605A). Triplex displacement assay determined DNA translocation activity in bulk. The translocation activity was studied by observing on an agarose gel the relative amount of triplex-forming oligonucleotide displaced by SpoIIIE. ATP hydrolysis assay showed a slower rate of ATP hydrolysis by mutant R605A than that of the wildtype. Triplex displacement assay showed an active mutant R605A subunit with the ability to displace triplex-forming oligonucleotides at a slower rate as compared to that ability of the wildtype.